Electroacupuncture Inhibits Atherosclerosis through Regulating Intestinal Flora and Host Metabolites in Rabbit.

METHODS: In this study, general rabbit conditions, vascular histology, metabolites, and intestinal flora structures were analyzed. Integrated analysis of metabolomics and 16S rRNA sequencing were performed. All the rabbits were randomly divided into four groups. The rabbit model of atherosclerosis was established. The histopathological change in the common carotid artery was assessed by HE staining and the structural change in the flora by 16S rRNA sequencing. HPLC-TOF-MS and Agilent MPP 12.1 were integrated to identify and screen out differential metabolites. Correlational analyses of every differential metabolite with intestinal flora were integrated on Omicshare platform. RESULTS: Atherosclerotic rabbits showed obvious changes in general conditions, significant fibrous cap and necrotic center on carotid artery, abnormal intestinal bacteria structure, and metabolites levels. Electroacupuncture improved the conditions, reduced lipid deposition on the carotid artery wall, diversified intestinal flora, and normalized host metabolism. Integrated analysis showed that 149 altered metabolites were related to 22 intestinal flora, among which eight intestinal floras and 21 metabolites have relationships with atherosclerosis. CONCLUSION: Electroacupuncture can effectively reverse atherosclerosis through manipulating the structural feature of intestinal flora to influence the host metabolites. The possible mechanisms involved activating signal pathways through host metabolites or affecting the activity of cardiovascular-related enzymes, or regulating host lipid metabolism directly.

The Beneficial Effects of Electroacupuncture at PC6 Acupoints (Neiguan) on Myocardial Ischemia in ASIC3 -/- mice.

This study aims to investigate the possible mechanisms of electroacupuncture (EA) at PC6 to improve myocardial ischemia (MI) by regulating the cardiac transient outward potassium current channel (Ito). According to the random number table, the mice were divided into six groups of six mice each: control group, MI group, PC6, LU7 (Lieque-point), ST36 (Zusanli-point), and nonacupoint group. Mice in the control group were injected with saline (20 mg/kg, 24 hours interval), and the other ASIC3 -/- mice were injected subcutaneously twice with isoproterenol (ISO) (20 mg/kg, 24 hours interval). In the preexperiment, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg of ISO were used, and the results showed that 5 mg/kg and 10 mg/kg of ISO both could induce acute MI, but shorter duration of sustained MI. On the other hand, an injection of 30 mg/kg can make the mice experience arrhythmia or die immediately, and EA was operated at PC6, LU7, ST36 acupoints, and nonacupoint in the mice of PC6, LU7, ST36, and nonacupoint groups, respectively, after injecting twice. Then Western blotting techniques (Western Blot) were used to analyze the protein expressions of Kv1.4, Kv4.2, Kv4.3, and KchIP2. The results of this experiment showed that the protein expressions of Kv1.4, Kv4.2, Kv4.3, and KChIP2 in MI group were significantly lower than those in the control group (p < 0.01). Compared with MI group, the results of PC6, LU7, and ST36 groups obviously increased (p < 0.05). Furthermore, the expressions of PC6 group were higher than LU7 group and ST36 group (p < 0.05). And electrocardiogram's T-waves showed obvious pathological changes in the MI group compared to the control group (p < 0.01). After EA, the abnormal T-waves voltage of ECG in PC6, LU7, and ST36 groups was improved (p < 0.05). In addition, the rate change of PC6 group was larger than that of both LU7 and ST36 groups (p < 0.05). But the T-waves voltage of the nonacupoint group was not significantly different than that of the MI group (p > 0.05).

[Effect of Electroacupuncture on ATP-sensitive Potassium Channel and Protein Kinase mRNA Ex- pression in Myocardial Ischemia Model Rats].

Objective To observe the effect of electroacupuncture (EA) at different acupoints on mRNA expressions of ATP-sensitive potassium channel (Kir6. 1, Kir6. 2) and conjugated protein (SUR2A, SUR2B) and protein kinases (PKA, PKG and PKC132) in myocardial ischemia model rats. Methods Myocardial ischemia model was established in healthy male SD rats via subcutaneously injec- ting ISO (85 mg/kg) multipointedly (medial root of limbs and the back). Then they were randomly divided into 4 groups, i.e., the model group, Neiguan (PC6) group, Lieque (LU7) group, non-acupoint group, 10 in each group. Besides, another 10 healthy rats were recruited as the control group. Corresponding EA was performed at respective acupoints to rats in Neiguan (PC6) group, Lieque (LU7) group, non-acu- point group, with dense-sparse wave, 2 -3 mA, 2 -20 Hz, needle retaining time of 20 min, once per day for 7 successive days. mRNA expression levels of Kir6. 1 and Kir6. 2, SUR2A, SUR2B, PKA, PKG, and PKCß2 in left ventricular myocardium were analyzed by Real-time PCR. Results Compared with the con- trol group, mRNA expressions of each index increased in the model group (P <0. 01). Compared with the model group, mRNA expressions of each index significantly decreased in Neiguan (PC6) group and Lieque (LU7) group (P<0. 01). Compared with Neiguan (PC6) group, mRNA expressions of each index significantly increased in Lieque (LU7) group and non-acupoint group (P <0. 01). Compared with Lieque (LU7) group, mRNA expressions of each index significantly increased in non-acupoint group (P <0. 05). Conclusion EA at Neiguan (PC6) could reverse mRNA expression changes of ATP-sensitive potassium channel (Kir6. 1 and Kir6. 2)and conjugated proteins (SUR2A and SUR2B) and protein kinases (PKA, PKG, and PKCß2).

[Effects of Electroacupuncture on Expression of Myocardial Chloride Channel-2 and CLCA Proteins in Mice with Acute Myocardial Ischemia].

OBJECTIVE: To observe the influence of electroacupuncture stimulation (EA) of "Neiguan" (PC 6)/"Lieque" (LU 7) on ST segment of electrocardiogram (ECG) and the expression of myocardiac chloride channel (CLC)-2 and calcium (Ca2+)-activated chloride channel accessory (CLCA) proteins in acute myocardial ischemia (AMI) mice, so as to explore its mechanisms underlying improvement of AMI. METHODS: Thirty ASIC 3-/- knock out mice were randomly divided into control, AMI model, EA-Neiguan (PC 6), EA-Lieque (LU 7) and EA-non-acupoint groups. The AMI model was induced by multiple subcuta-neous injection of isoprenaline (ISO, 0.5 g/L, 20 mg/kg). EA was applied to bilateral PC 6, LU 7, or non-acupoint[the mid-point between "Tianshu" (ST 25) and "Shenque" (CV 8)] for 20 min, once daily for 7 days. The ST segment of ECG of the standard Ⅱlimb-lead was recorded using a PowerLab data acquisition device, the change of myocardial histology observed by using microscope after HE staining, the activity of serum superoxide dismutase (SOD) detected using Colorimetric method, and the expression of CLC-2 and CLCA proteins of the left ventricle myocardium detected by Western blot. RESULTS: Outcomes of HE staining showed that the ischemic injury (sarcoplasm swelling and necrosis, etc.) of the left ventrical myocardial tissue after mo-deling was relatively milder in the EA-PC 6 group, not in the EA-non-acupoint group. The SOD activity was significantly lower in the model group than in the control group (P<0.01), and obviously increased in the EA-PC 6 group(P<0.01), but not in the EA-non-acupoint group (P>0.05). The expression levels of myocardial CLC-2 and CLCA proteins were significantly up-regulated in the model group compared with the control group (P<0.01) and markedly down-regulated in the EA-PC 6 group (not in the EA-non-acupoint group) in comparison with the model group(P<0.01), suggesting a specificity of effects of EA-PC 6 in improving myocardial injury and down-regulating CLC-2 and CLCA protein expression. CONCLUSIONS: EA of "Neiguan" (PC 6) can effectively suppress the increase of expression of CLC-2 and CLCA proteins in the left ventricle myocardium, which may contribute to its effect in relieving myocardial injury in mice.

[The Impact of Electroacupuncture Intervention on Expression of 5-HTR 1 B/2 C Genes in Mice under Radiation Stimulation from Mobile Phone].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Yifen" (TE 17), "Shenshu" (BL 23) on the expression of 5-hydroxytryptamine receptor 1 B (5-HTR 1 B) mRNA and 5-hydroxytryptamine receptor 2 C (5-HTR 2 C) mRNA in the cochlear nucleus tissue in mice experiencing radiation from mobile phone, so as to explore its mechanisms underlying improvement of tinnitus. METHODS: Thirty Kunming mice were randomly divided into control group (n = 6) and modeling group (n = 24). The tinnitus model was established by giving the mice with mobile phone-radiation for 1 h in the morning and 1 h in the afternoon, continuously for 40 days. EA stimulation was applied to "Yifeng" (TE 17) group (n = 6) and "Shenshu" (BL 23) group (n = 6) for 20 min, once a day for 7 days. The expression of 5-THR 1 B/2 C mRNA in the cochlear nucleus was assayed by fluorescence quantitative polymerase chain reaction (real time-PCR). RESULTS: The expression level of 5-HTR 1 B was significantly lower in the model group than in the control group (P < 0.05), while that of 5-HTR 2 C mRNA significantly increased (P < 0.01). TE 17 group received a significant acupoint intervention effect (P < 0.01). Compared with TE 17 group, BL 23 group received a weaker effect (P < 0.05). CONCLUSION: EA of TE 17 can up-regulate expression level of 5-HTR 1 B and down-regulate expression level of 5-HTR 2 C in the cochlear nucleus in mice experiencing mobile-phone radiation.

[Effect of electroacupuncture stimulation of "Neiguan" (PC 6) on expression of alpha- and beta-subunit proteins of voltage-gated sodium channels in rats with myocardial ischemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Neiguan" (PC 6) on expression of sodium (Nav) channel α-subunit 1.5 and Nav ß-subunits ß 1-ß 4 (the known myocardial sodium channel proteins) in acute myocardial ischemia (AMI) rats so as to explore its mechanisms underlying protection of ischemic myocardium. METHODS: Sixty SD male rats were randomly divided into normal control, AMI model, Neiguan (PC 6), Lieque (LU 7) and non-acupoint groups. The AMI model was established by intravenous injection of Isoprenaline (85 mg/kg) once daily for 2 days. Myocardial Nav 1.5 and Nav ß 1, ß 2, ß 3, ß 4 protein expression levels were detected by Western blot. RESULTS: In comparison with the control group, myocardial Nav 1.5, ß 1, ß 2, ß 3 and ß 4 protein expression levels were significantly down-regulated in the AMI model group (P<0.01). After EA stimulation, compared with the model group, the expression levels of Nav 1.5, Nav ß 1, ß 2, ß 3 and ß 4 protein expression levels were significantly up-regulated in the Neiguang (PC 6) and Lieque (LU 7) groups (P<0.01) rather than in the non-acupoint group (P>0.05). There was no statistical significance between the Neiguan (PC 6) and Lieque (LU 7) groups in ß 4 protein expression level (P>0.05). CONCLUSION: EA stimulation of both PC 6 and LU 7 can significantly reverse AMI-induced down-regulation of myocardial Nav 1.5, ß 1, ß 2, ß 3 and ß 4 protein expression levels in AMI rats, which might contribute to its function in improving AMI by reducing calcium overload.

[Effect of Electroacupuncture Stimulation of "Neiguan" (PC 6) on Expression of Myocardial Chloride Channel-related Genes, PKC and PKG Proteins in Myocardial Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Neiguan" (PC 6), etc. on expression levels of myocardial chloride (CL-) channel-related genes and intracellular protein kinase C (PKC) protein in myocardial ischemia (M) rats. METHODS: Seventy SD rats were randomly divided into control group (n = 10), model group (n = 15) , Neiguan (PC 6) group (n = 15), Lieque (LU 7) group (n = 15) and non-acupoint group (n = 15). The MI model was established by i. p. of isoproterenol (ISO, a sympathomimetic beta adrenergic agonist). Electroacupuncture stimulation was applied to bilateral "Neiguan" (PC 6), "Lieque" (LU 7), or non-acupoint [the mid-point between "Tianshu" (ST 25) and "Shenque" (CV 8)] for 15 min, once a day for 7 days. Quantitative RT-PCR was employed to detect the expression levels of cystic fibrosis transmembrane conductance regulator (CFTR, a CL-channel) mRNA and chloride channel calcium-activated 1 (CLCa 1, a member of the family of calcium-activated chloride channels, CLCa) mRNA in the left cardiac ventricle tissue, and Western blot was used to detect the expression level of myocardial PKC protein of the left ventricle. RESULTS: Compared with the control group, the expression levels of myocardial PKC protein, and CLCa 1 and CFTR genes were significantly increased in the model group (P<0.05). In comparison with the model group, the expression levels of myocardial PKC protein, and CFTR mRNA and CLCa 1 mRNA in the Neiguan group, and PKC protein and CLCa 1 mRNA in the Lieque and non-acupoint groups, as well as CFTR mRNA in the Lieque group were notably down-regulated (P<0.05). No significant change was found in the expression of CFTR mRNA in the non-acupoint group (P>0.05), and no significant differences were found between Neiguan and Lieque groups in the expression levels of PKC protein (P>0.05). The effects of "Neiguan" (PC 6) were obviously superior to those of non-acupoint in down-regulating myocardial PKC protein, CLCa 1 mRNA and CFTR mRNA (P<0.05). CONCLUSION: EA stimulation of "Neiguan" (PC 6) can down-regulate the expression of myocardial PKC protein, CFTR and CLCa 1 genes in Ml rats, which may contribute to its effect in protecting rnyocardium from ischemic injury.

The Beneficial Effects of Electro-acupuncture at PC6 (Neiguan-point) of Gene and Protein Expressions of Classical Inward-rectifier Potassium Channels in Myocardial Ischemic Rats.

This study is aim to investigate the effect of electro-acupuncture at PC6 (Neiguan-point) on the gene and protein expressions of classical inward-rectifier potassium channels (Kir) in myocardial ischemia (MI) rats induced by isoproterenol (ISO). With ten for each one, 50 rats were divided into 5 groups which were control group, MI group, PC6 group, LU7 (Lieque-point) group and non-acupoint group. The control group was injected normal saline solution (85 mg/kg), the other groups were injected ISO (85 mg/kg). All the rats were injected once daily for two days and recorded electrocardiograms (ECGs) after every injection. Electro-acupuncture (EA) was operated at PC6, LU7 and non-acupoint respectively in the rats of PC6 group, LU7 group and non-acupoint group after twice injections. EA was performed to these three groups with disperse-dense wave (4-20 Hz), pulse amplitude of 14V, 20 mins a day remaining 7 days. The gene and protein expressions of Kir2.1, Kir2.2 and Kir2.3 were analyzed by Western Immunoblotting Technology (Western Blot) and Real-time Fluorescence Quantitative Polymerase Chain Reaction (RT-PCR). But it is regrettable that we did not detect meaningful gene and protein expressions Kir2.3, and the expressions of Kir2.1 and Kir2.2 in MI induced groups were lower [The gene and protein decreased 39.4 ± 27.3% and 38.7 ± 17.1% respectively.] than control group (P < 0.05). Compared with MI group, the results of PC6 group and LU7 group increased [PC6 group: the gene and protein increased 42.9 25.0% and 42.2 ± 10.0% respectively. LU7 group: the gene and protein increased 23.8 ± 50.1% and 21.1 ± 32.5% respectively.] obviously (P < 0.05) after EA, furthermore the expressions of PC6 group were higher [The gene and protein increased 15.4 ± 16.7% and 17.3 ± 60% respectively.] than LU7 group (P < 0.05). The results show that PC6 has a better positive effect than LU7 on MI rats, and the mechanism is probably that EA at PC6 can significantly increase the gene and protein expressions of Kir2.1 and Kir2.2.

Effects of acupuncture on the tissue distribution of Paclitaxel in lung carcinoma mice.

OBJECTIVE: To study whether acupuncture affects the tissue distribution of Paclitaxel in mouse lung carcinoma. METHODS: Totally 90 mice were divided into Paclitaxel group, Paclitaxel + Feishu (BL13) group, and Paclitaxel + Lingtai (DU10) group. Each group was consisted of 30 mice. After Paclitaxel injection, the mice received electro-acupuncture at Feishu or Lingtai acupoints once a day for 8 days. The effect of acupuncture on the tissue distribution of Paclitaxel was evaluated using high-performance liquid chromatography at 1, 2, and 3 h, respectively. The lung, liver, spleen, and kidney were examined for the concentration of Paclitaxel seperately. RESULTS: Paclitaxel was widely distributed in various organs, particularly in the lung, liver, and kidney. Acupuncture at Lingtai or Feishu acupoints resulted in an obvious decrease of Paclitaxel distribution in kidney and delayed Paclitaxel distribution in liver. Meanwhile, it increased the time of metabolism. Acupuncture at Feishu acupoint facilitated the delivery of Paclitaxel to lung more effectively than did acupuncture at Lingtai acupoint. CONCLUSIONS: Applying acupuncture at particular acupoints can influence tissue distribution of Paclitaxel. Tissue distribution change might be one of the mechanisms of acupuncture treatment during chemotherapy.

[Effects of acupuncture on distribution taxis of paclitaxel in mice with lung cancer].

OBJECTIVE: To observe the effects of acupuncture at "Feishu" (BL 13) and "Lingtai" (GV 10) on distribution taxis of paclitaxel in mice with lung cancer to discuss targeted relationship between acupoints and corresponding viscera. METHODS: According to randomized digital table, 315 SPF-grade BALB/C female mice were divided into 7 groups: blank group (group A), model group (group B), medication group (group C), acupuncture at non-acupoint group (group D), acupuncture at Feishu group (group E), acupuncture at Lingtai group (group F) and acupuncture at Feishu and Lingtai group (group G), 45 mice in each one. Except the blank group, the remaining groups were treated with N-nitroso-tris-chloroethyl urea (NTCU) to establish the model of squamous-cell carcinoma. After model establishment, group A, group B and group C were not treated with acupuncture; group A and group B were treated with intraperitoneal injection of 0.9% sodium chlorvde solution by 6 mL/kg while group C was treated with intraperitoneal injection of paclitaxel by 6 mL/kg. The group D, group E, group F and group G were treated with acupuncture at non-acupoint, "Feishu" (BL 13), "Lingtai" (GV 10) and "Feishu" (BL 13) plus "Lingtai" (GV 10), respectively, then were intraperitoneally injected with paclitaxel by 6 mL/kg. The treatment was all given once a day for continuous 10 days. 15 min, 30 min, 1 h, 2 h, 8 h, 12 h and 24 h after the treatments, 6 mice in each group were randomly selected and sacrificed to collect samples of lung, liver, spleen, kidney and heart, etc. High performance liquid chromatography was applied to measure the concentration of paclitaxel in each organ (lung, liver, spleen, kidney and heart) at different time points. RESULTS: (1) The content of paclitaxel in lung, kidney and heart reached the peak at 2 h, then decreased significantly in group C, group D, group E, group F and group G; the content of paclitaxel in spleen showed downtrend at each time point. The content of paclitaxel in liver reached the peak at 2 h in group C and group D; the content of paclitaxel reached the peak at 8 h in group E, group F and group G. (2) The content of paclitaxel in lung in group E and group G was higher than that in group C and group D at each time point (all P < 0.01); the content of paclitaxel in lung in group F was higher than that in group C (P < 0.01) and group D (P < 0.01) only at time point of 2 h. The content of paclitaxel in lung in group G was higher than that in group F at each time point (all P < 0.05). There was no statistical difference between group G and group E (all P > 0.05). CONCLUSION: Acupuncture at "Feishu" (BL 13) and "Lingtai" (GV 10) could influ- ence the metabolism of paclitaxel in lung-cancer mice, leading to distribution change in each organ. As a result, it could cause targeting effects, which is more significant at "Feishu" (BL 13) and "Lingtai" (GV 10).